What complications would you expect to see if a patient is exposed to chlorine chloramines?

Chlorine, Chloramines, Hydrochloric Acid, and Related Chemicals

Among toxicants causing pulmonary responses,chlorine is a common and potent irritant inhalant accounting for substantial human morbidity.17,87,88 Common forms of exposure include industrial leaks,89,90 environmental releases primarily during transport,91,92 water purification,93 swimming pool–related events,94,95 household cleaning product misadventures,96 and even homemade chemical bombs97 or intentional terrorist use.98

In the form of a yellow-green acrid gas, industrial and environmental releases typically have clear-cut exposure histories. Because the gas is heavier than air, higher contamination can be expected in low-lying areas (hence, its use in trench warfare in World War I).99 Other environmental conditions, however, may supervene. Examples include one well-documented case of the gas rising along the heated outside wall of a factory where rooftop workers were exposed.89 In another case, chlorine initially collected in a basement but then was sucked up into the central heating system of a dormitory.100

The history of exposure to chlorine may be less straightforward when the chlorine is generated after de novo generation from chlorine-containing products. Chlorine gas can be generated from liquid bleach containing hypochlorite or from dry powdered bleach containing chlorinated phosphate. In either liquid or powdered bleaches, the chlorine gas is liberated on contact with acids, which can be common in other household products (such as those containing muriatic [i.e., hydrochloric], phosphoric, or hydrofluoric acid96) or in industrial settings.53 In contrast, mixing chlorine-containing products with ammonia leads to release of chloramines (monochloramine [NH2Cl] and dichloramine [NHCl2]) and related chemicals, especially nitrogen trichloride (NCl3), chemicals whose irritant effects are attributed to in situ pulmonary reactions releasing chlorine, hypochlorous acid, and ammonia.96,101,102 In aquatic venues, including swimming pools, inadvertent mixing of the chlorinated water with nitrogen donors can also happen, with potential irritant effects attributed to nitrogen trichloride in particular.101,103 A report of a public aquatic venue mishap found that inadvertent mixing of sodium hypochlorite and hydrochloric acid led to toxic chlorine gas release resulting in a constellation of adverse health effects, including vomiting, coughing, and eye irritation, among several dozen affected bathers.104 Chloramines evolved from mixing chlorine and ammonia should not be confused with Chloramine-T (sodium-N-chlorine-p-toluene sulfonamide), a disinfectant that can act as a chemical sensitizer leading to allergic asthma and other anamnestic responses.105

6. IODINATION METHODS

Iodine can be substituted onto the aromatic side-chain of tyrosine residues to yield a stable compound which is a highly efficient tracer. Iodine may also substitute onto other amino acids such as histidine and phenylalanine, but the rate of the latter reaction is 30–80 times less than that for tyrosine. The precise nature and location of the substitution onto tyrosine varies with specific activity, the nature of the peptide molecule, and the method for iodination. At low levels of specific activity (one atom of iodine per molecule or less) most substitutions are single (i.e., mono-iodotyrosine); at higher levels of activity diiodotyrosine may be formed. In a given peptide molecule, tyrosine residues may differ widely in their accessibility. Thus, insulin has four tyrosines, in positions 14 and 19 on the A chain, and 16 and 26 on the B chain; most iodine substitution occurs at A14, some at A19, and very few at B16 or B26 (4).

The so-called “direct” methods have in common the conversion of iodide (I−), which is relatively unreactive, into a more reactive species such as free iodine (I2), or positively charged iodine radicals (I+). The basic chemistry of this reaction is poorly understood. The “indirect” methods involve conjugation of ligand to a molecule already labeled with iodine.

6.4. Conjugation Labeling

For this procedure a carrier molecule is used: the carrier incorporates a phenol or imidazole group which can be iodinated, and an amine group which can be coupled directly to carboxyl groups on the ligand or its derivatives (11) (Figs. 12.2 and 12.3). There are also carriers for conjugation to amine groups on the ligand (e.g., fluorescein isothiocyanate, N-acetyl-1-histidine). The carrier may be iodinated either before or after attachment to the ligand.

FIGURE 12.2. Compounds which can be used as a “handle” for the indirect attachment of 125I to the ligand.

(Modified from Chard, 1990.)Copyright © 1990

FIGURE 12.3. Schematic of the conjugation labeling technique.

(Modified from Chard, 1990.)Copyright © 1990

Conjugation labeling has several advantages over direct iodination: it is less damaging to the ligand, it can be applied to peptides without tyrosine residues, the final reaction (mixing of the iodinated ester and the peptide) is very simple, and it can be applied to nonpeptide materials (e.g., steroid hormones) which cannot be iodinated directly. The disadvantages include: (i) the substituted label is considerably larger than the iodine atom and may lead to physicochemical alteration of the tracer; (ii) with haptens such as steroids and drugs, the tracer may bind well to antibody but fail to be displaced by unlabeled material, i.e., a “flat” standard curve; this occurs because the antiserum contains populations of high-affinity antibodies directed toward the bridge between the hapten and the tag (Fig. 12.4). Sometimes this problem can be solved by selection of a particular antiserum (12), by using disequilibrium conditions, or by choosing different bridges or bridge sites for the immunogen and the tracer (13, 14). As a general rule, the steric bulk of the tracer bridge should be greater than that of the immunogen (e.g., in a progesterone assay, an 11-hemisuccinate conjugate should be used for immunisation and an 11-glucuronide conjugate for labeling (15); (iii) iodotyrosine or iodohistamine can itself bind to serum proteins, especially thyroxine-binding globulin, and thus produce artifacts in unextracted samples (16).

FIGURE 12.4. A problem with the conjugation labeling of small molecules (haptens). The antiserum will contain populations of antibodies directed to the carrier protein (A), the hapten (C), and the bridge between the two (B). If the tracer contains the same bridge, then it will be firmly bound by antibodies to the bridge and cannot be displaced by pure hapten (i.e., standard or endogenous material). Greatest sensitivity and slope are achieved when the bridge is dissimilar to that in the immunogen, but unfortunately specificity is often best when the label and immunogen are similar.

(Modified from Chard, 1990.)Copyright © 1990

Water and Water Treatment

A standard 4-hour HD session exposes the patient to 120 to 160 liters of water. Therefore water quality is of paramount importance to the patient's well-being. A typical water purification plant is shown inFig. 93.2. Water from municipal sources is filtered to remove particulate matter. Activated carbon adsorbs substances such as endotoxins, chlorine, and chloramines. Downstream water softeners use a resin coated with sodium ions, which are exchanged for calcium and magnesium ions before the water enters the reverse osmosis (RO) system. During RO, water is pumped at high pressure (15 to 20 bar) through a tight membrane. The small pore size of these membranes (0.5 to 0.5 nm) provides an absolute barrier for molecules larger than 100 to 300 d. This process rejects over 99% of all bacteria, viruses, pyrogens, and organic materials. Optional ultraviolet irradiation upstream of a filter is used to further protect against contamination with bacteria.

Standards for chemical quality of water are widely accepted, but there is less consensus regarding acceptable levels of bacterial and endotoxin contamination. The microbiologic standards for HD water, dialysis fluid, and substitution fluid vary across countries (Table 93.2).3 Endotoxin concentrations below 0.25 endotoxin units (EU)/ml are suggested, and many support the use of 0.06 EU/ml or lower. Municipal water supplies may contain a variety of contaminants that are toxic to HD patients. Substances added to the water supply such as aluminum and chloramines cause significant morbidity. Aluminum accumulation may result in a severe neurologic disorder, bone disease, and erythropoietin-resistant anemia. Is it recommended to measure plasma aluminum concentration regularly in situations in which aluminum exposure of the patient is likely to occur. Levels should be below 1 µmol/l, and levels above 2 µmol/l should prompt the search for excessive exposure. Chloramines have been associated with hemolysis and methemoglobinemia. Copper and zinc may leach from plumbing components and may cause hemolysis. Lead has been associated with abdominal pain and muscle weakness. Nitrate and nitrite may cause nausea and seizures. High concentrations of calcium may cause the hard water syndrome, characterized by acute hypercalcemia and hypomagnesemia, hemodynamic instability, nausea, vomiting, muscle weakness, and somnolence.

Gram-negative bacteria produce endotoxins, and fragments of these endotoxins may be responsible for some dialysis-related symptoms. Exposure to bacteria and endotoxin is associated with rigors, hypotension, and fever. Low levels of microbiologic contaminants may be a major cause of chronic inflammation in HD patients. Use of a polysulfone or polyamide filter in the dialysate line may be adequate to remove endotoxins, but smaller molecules, including bacterial DNA fragments, may pass through the dialyzer and stimulate immune cells. In the absence of routine hot water disinfection of the machine and the connections to the water loop, the only way endotoxin concentration can be kept low is by frequent measurement and disinfection of the entire system when concentration exceeds accepted standards. Liquid bicarbonate dialysis fluid concentrate distributed in a central distribution system may be a source of bacterial growth and should be replaced daily; acid concentrates in canisters and bicarbonate powder represent no bacterial growth risk.

10.4.6 The ‘Iodo-beads’ Method

Chloramine-T, covalently attached to polystyrene beads, has been shown to be an efficient and gentle reagent for protein iodination (Markwell, 1982). The beads are available from Pierce Chemicals under the name ‘Iodobeads’. The reaction is simple and efficient.

On the day of use, Iodo-beads are washed twice in PBS, and blotted dry on filter paper. Then, 100 μg protein in 500 μl PBS is added, followed by 1 mCi of Na125I. The iodination reaction is initiated by addition of one or more Iodo-beads, and allowed to proceed for 15 min at room temperature. The reaction is terminated by removal of the liquid and gel filtration to remove unreacted iodide.

The reaction volume is not critical, and shaking is evidently not necessary. The reaction is not inhibited by SDS, 8M urea, or 2% Nonidet P-40, although it is abolished by reducing agents such as mercaptoethanol. The efficiency of incorporation was 35% with one bead, and virtually 100% with five beads.

Water Treatment

Because HD patients are exposed to as much as 600 L of dialysate water/week, treating the water used to generate dialysate is essential to avoid exposure to harmful substances such as aluminum, chloramines, fluoride, endotoxins, and bacteria.324–328 Technical advances such as high-flux dialyzers, reuse or reprocessing of dialyzers, and bicarbonate-based dialysate have made high water quality even more imperative. To avoid these complications, tap water is softened, exposed to charcoal to remove contaminants such as chloramine, filtered to remove particulate matter, and then filtered again under high pressure (reverse osmosis) to remove other dissolved contaminants (Fig. 63.18). A complete review of this topic is beyond the scope of this chapter, and readers are referred to reviews on the topic.324–329 Highlights are discussed later.

Therapie

Iodination of pYT22

Peptide is iodinated using chloramine-T (8). A 5-μg aliquot of pYT22 is redissolved in 10 μl 0.5 M sodium phosphate buffer, pH 7.5. Under an appropriate containment hood 1 mCi of Nal25I (5 μl) is added, followed by 10 μl of 1 mg/ml chloramine-T in water; the chloramine-T should be dissolved less than 5 min before addition to the peptide. Following vigorous mixing the reaction is allowed to proceed for 1 min before quenching with 10 μl of 2 mg/ml sodium metabisulfite in water. The resulting iodination is immediately diluted with 100 μl 0.1% trifluoroacetic acid (TFA) containing 1% Polypep (Sigma). The Polypep minimizes adsorption losses and removes remaining free iodine while having little detrimental effect on the HPLC column (see below). Essentially similar methods are used for the iodination of 125I-labeled pYE27.

INHALATION

Inhalation of chlorine or chloramine gas can often be managed on-site or at home with fresh air, cool oral liquids, and inhaled steam. Most patients with exposure to chlorine or chloramine gas will have resolution of symptoms within 6 hours of exposure. If symptoms worsen at any time or persist for longer than 6 hours, emergency supportive care is warranted.39 Patients with preexisting respiratory conditions may be more sensitive to exposure, but can still be monitored at home if symptoms are mild and air exchange is not compromised.31 Anecdotal evidence has suggested that nebulized sodium bicarbonate may be an effective adjunct for chlorine gas exposure. Prospective, randomized, controlled trials have not been carried out. A nonrandomized, nonblinded investigation of its use in chloramine gas exposure did not find a significant difference compared with oxygen alone.32 Inhalation of powdered oxygen bleach can be managed with fresh air and cool oral fluids. If symptoms persist for more than 6 hours after inhalation, emergency supportive care should be sought.

Concluding Remarks

Postlabeling reduction of chloramine-T prepared 125I-labeled [Tyr8]SP with mercaptoethanol yielded a 125I-labeled [Tyr8]SP derivative which significantly improved the radioimmunoassay for SP. It presumably consists of the pure labeled thioether. Therefore, the quantification of neuropeptide-like immuno-reactivities, such as SPLI, could be achieved even in small sample volumes of biological fluids (e.g. CSF) in which the peptide was present in the lower picogram per milliliter range.

It is a well-known fact that CSF represents a unique compartment for the in vivo study of biologically active substances and their metabolites in the CNS (53). Lumbar fluid is easily accessible for measurement, whereby sample volumes greater than 10 ml are usually obtained. However, the ventricular space represents a more appropriate compartment for the quantification of CNS SPLI as described for both somatostatin-like immunoreactivity (SLI) (54–56) and SPLI (45). This is due to the proximity of the ventricles to adjacent brain structures from which the peptide is released. Therefore, the measurement of SPLI of this origin will provide more insight into the metabolism of SP than measurement of lumbar CSF. However, ventricular CSF is available only in exceptional cases after stereotactic surgery with ventriculography (57). In general, analytical investigations of sample constituents can only be performed with volumes far below 5 ml. Therefore, extremely sensitive methods are required to determine the low amounts of ventricular SPLI by aid of the HPLC-RIA coupling method.

Additionally it is evident that the splitting of SPLI into a variety of structurally related peptides will cause a decrease of individual concentrations. For this reason it may be much more reasonable in routine determinations of SPLI in small CSF volumes of about 1–2 ml (e.g., in lumbar CSF) to focus only on immunoreactivity coeluting with SP(1–11). It seems to be reasonable that different undecapeptide concentrations may reflect individual alterations in the synthesis and/or the metabolism of SP precursors. Therefore changes in SP(1–11) concentrations in the CSF of patients with well-established neurological disorders should be monitored and correlated to the pathobiochemical process underlying the disease.

The preparation of 125I-labeled [Tyr8]SP, which contains an intact Met11 residue for use in RIA experiments, represents an appropriate tool to reach a level of high sensitivity for detection and quantification of SPLI in biological fluid systems. In conclusion, additional attempts are required to preserve the Met11 from oxidation during iodination of SP. This aim should be achieved by application of the lactoperoxidase method after appropriate optimization of the reaction conditions (e.g., variation of buffer strength and/or pH). In this way, a convenient “one-pot” reaction would allow the rapid preparation of a radiolabel with both high specific activity and selectivity toward an antibody against synthetic SP.

5.6.3.1 Catalytic Aziridination with Chloramine-T

Albone et al. used commercially available chloramine-T trihydrate (36·3H2O, Scheme 24) as a nitrene source for the catalytic aziridination of several aryl alkenes by a Cu complex of pyridine-based bidentate nitrogen ligand.52 Up to 76% yield was obtained with Cu/36-based aziridination. Using 1  mol% of a Fe(III) corrole complex as the catalyst, Simkhovich and Gross also reported that chloramine-T trihydrate could be used for catalytic aziridination of aromatic alkenes in 48–60% yields.53 Comparison studies indicated chloramine-T is a more practical nitrene source than PhI=NTs.